Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. The stationary phase of HILIC is a polar and hydrophilic phase which results in enhanced retention for polar analytes. The mobile phase of HILIC is a reversed-phase type high organic eluent, for example, a mixture of water and acetonitrile. A mechanism of separating analytes in HILIC can be a combination of partitioning, ion exchange and reverse-phase chromatography.
Prior to the present invention, HILIC has seen limited use for the separation and analysis of large proteinaceuous biomolecules, such as glycoproteins. The authors of T. Tetaz et al., J. Chomatogr., A 1218, 5892-5896 (2011), explored the analysis of intact soluble proteins using HILIC with mediocre results, presenting chromatograms indicative of relatively low resolution separations. Similarly, the authors of, “Separations of Intact Glycoproteins by HILIC” at the 33rd International Symposium and Exhibit on the Separation and Characterization of Biologically Important Molecules, Jul. 17-19, 2013 in Boston, Mass., USA, expressed that glycoprotein separations by HILIC would be of significant interest though at present this poses a significant challenge. Their presented work was limited to separation of small (<20 kDa) glycoproteins.
Moreover, Guillarme (“What You Need to Know About HILIC” Jul. 1, 2013 LCGC NORTH AMERICA Volume 31, Issue 7, pp. 560-563) notes that the peak shape of large proteins (greater than 20 kDa) using HILIC may be unacceptable as, prior to the present invention, optimized wide-pore HILIC phases are not available.
There remains a need for HILIC materials designed for high efficiency/high resolution separations of large biomolecules (e.g. larger average pore diameters) along with improved chromatographic methodologies for the analysis of such samples, including those containing proteinaceous biomolecules modified with polar groups known to those skilled in the art as glycans.